Vectors based on lytic viruses such as polyoma have been used for short-term expression, but tend to be unstable, and replicate many times per cell cycle (Lebkowski, J. S., et al., MOL. CELL. BIOL. 4:1951-1960, 1984). Vectors based on bovine papilloma virus have also been developed but do not consistently replicate once per cell cycle (Gilbert, D. M., et al., CELL 50:59-68, 1987; Ravnan, J.-B., et al., J. VIROL. 66:6946-6952, 1992). Further bovine papilloma virus-based vectors show a high frequency of rearrangements (Ashman, C. R., et al., SOMATIC CELL MOL. GENET. 11:499-504, 1985; DuBridge, R. B., et al., MOL. CELL. BIOL. 7:379-387, 1987).
In human and primate cells, vectors based on Epstein-Barr virus (EBV) have been developed (Yates, J., et al., PROC. NATL. ACAD. Sci. USA. 81:3806-3810, 1984; Reisman, D., et al., MOL. CELL. BIOL. 5:1822-1832, 1985; Lupton, S., et al., MOL. CELL. BIOL. 5:2533-2542, 1985). These vectors typically replicate once per cell cycle (Adams, A., J. VIROL. 61:1743-1746, 1987; Yates, J. L., et al., J. VIROLOGY 65:483-488, 1991; Haase, S. B., et al., NUC. ACIDS RES. 19:5053-5058, 1991) and are stably maintained over the long-term with a low mutation frequency (DuBridge, R. B., et al., MOL. CELL. BIOL. 7:379-387, 1987; DuBridge, R. B., et al., MUTAGENESIS 3:1-9, 1988; Drinkwater, N. R., et al., PROC. NATL. ACAD. SCI. USA 83: 3402-3406, 1986). These vectors have been used for cloning and expression studies in human and simian cells (Margolskee, R. F., et al., MOL. CELL. BIOL. 8:2837-2847, 1988; Young, J. M., et al., GENE 62:171-185, 1988; Belt, P. B. G. M., et al., GENE 84:407-417, 1989; Peterson, C., et al., GENE 107:279-284, 1991). Stable transformation frequencies are high because integration into the genome is not required, and recovery of cloned sequences is achieved by plasmid extraction. However, rodent cells are not permissive for EBV replication, and no rodent counterpart of EBV has been described (Yates, J. L., et al., NATURE (LONDON) 313:812-815, 1985).
U.S. Pat. No. 4,959,317 (Sauer, et al.) discloses the use of Cre-Lox site-specific recombination to achieve gene transfer in eukaryotic cells. However, the system described does not provide efficient or stable integration of transferred DNA into the host genome (see e.g., (Sauer, et al., (1993) Methods in Enzymology 225: page 898). This is largely due to the fact that excision of transferred DNA out of the genome, by way of intramolecular exchange, predominates over integration of DNA into the genome, by way of intermolecular site-specific recombination.
U.S. Pat. No. 5,928,914 (Leboulch, et al.) describes methods and compositions for transforming cells, resulting in efficient and stable site-specific integration of transgenes. Transformation is achieved by introducing into a cell an acceptor vector, preferably a retroviral vector, which integrates into the genome of the cell. The acceptor vector comprises two incompatible lox sequences, L1 and L2. A donor vector is then introduced into the cell comprising a transgene flanked by the same L1 and L2 sequences. Stable gene transfer is initiated by contacting the lox L1 and L2 sequences with Cre recombinase.